Testing Host-Plant Driven Speciation in Phytophagous Insects : a Phylogenetic Perspective

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1 Testing host-plant driven speciation in phytophagous insects : a phylogenetic perspective

3 Emmanuelle Jousselin* 1 , Marianne Elias 2

4 1. CBGP, INRA, CIRAD, IRD, Montpellier SupAgro, Univ Montpellier, Montpellier, France

5 2 Institut Systématique Evolution Biodiversité (ISYEB), Muséum national d'Histoire naturelle, 6 CNRS, Sorbonne Université, EPHE, Université des Antilles, 57 rue Cuvier, CP 50, 75005 Paris, 7 France.

8 *corresponding author : [email protected]

10 Keyword: coevolution, herbivory; host-plant specialization, phylogeny, speciation.

12 Abstract

13 During the last two decades, ecological speciation has been a major research theme in 14 evolutionary biology. Ecological speciation occurs when reproductive isolation between 15 populations evolves as a result of niche differentiation. Phytophagous insects represent model 16 systems for the study of this evolutionary process. The host-plants on which these insects feed and 17 often spend parts of their life cycle constitute ideal agents of divergent selection for these 18 organisms. Adaptation to feeding on different host-plant species can potentially lead to ecological 19 specialization of populations and subsequent speciation. This process is thought to have given 20 birth to the astonishing diversity of phytophagous insects and is often put forward in 21 macroevolutionary scenarios of insect diversification. Consequently, numerous phylogenetic 22 studies on phytophagous insects have aimed at testing whether speciation driven by host-plant 23 adaptation is the main pathway for the diversification of the groups under investigation. The 24 increasing availability of comprehensive and well-resolved phylogenies and the recent 25 developments in phylogenetic comparative methods are offering an unprecedented opportunity to 26 test hypotheses on insect diversification at a macroevolutionary scale, in a robust phylogenetic 27 framework. Our purpose here is to review the contribution of phylogenetic analyses to investigate 28 the importance of plant-mediated speciation in the diversification of phytophagous insects and to 29 present suggestions for future developments in this field.

31 Introduction

32 The idea according to which new species arise through adaption to different ecological 33 niches constitutes the core of Darwin’s work. This process is now termed ecological speciation 34 and its study has become an intense field of research in evolutionary biology (Nosil 2012; Nosil et 35 al. 2002; Rundle & Nosil 2005; Schluter 2009). Phytophagous insects*1 have always been at the 36 forefront of these investigations (Drès & Mallet 2002; Elias et al. 2012; Forbes et al. 2017; Funk 37 et al. 2002; Matsubayashi et al. 2010). The hypothesis of ecological speciation resulting from 38 divergent selection exerted by host-plants was put forward a long time ago to explain the 39 formation of new species of insects (Brues 1924; Walsh 1864). There are several model systems 40 on which this scenario has been explored. One text-book example of host-plant driven incipient 41 speciation is the apple maggot (Rhagoletis pomonella complex) in which the evolution of new 42 feeding preferences on the recently introduced domesticated apple (Malus pumila) has supposedly 43 led to the emergence of specialized host races (Berlocher 2000; Bush 1975; Powell et al. 2014). 44 Stick insects, leaf beetles (Nosil et al. 2012; Rundle et al. 2000), butterflies (McBride & Singer 45 2010), and the pea aphid also star among model systems in the study of host-driven speciation 46 (Via et al. 2000; Caillaud & Via 2000; Peccoud et al. 2009; Smadja et al. 2012). In all these 47 examples, the speciation scenario hypothesizes that: 1) the restricted utilization of distinct sets of 48 host-plant species by insect populations is the result of adaptive trade-offs; 2) hybrids with 49 intermediate phenotypes (in terms of traits involved in host-plant adaptation*) fare poorly on 50 parental host-plants and therefore gene flow between populations is reduced; 3) gene flow can be 51 further reduced through the evolution of assortative mating, especially when host-plants also 52 represent mating sites. In support of this scenario, many studies show the existence of genetically 53 differentiated host races in insect species. Some studies have quantified selection against hybrids 54 (McBride & Singer 2010; Gow et al. 2007) and some studies have uncovered genomic regions 55 that determine host-plant preference and performance on alternative hosts (Egan et al. 2008; 56 Smadja et al. 2012; Soria-Carrasco et al. 2014). 57 The role of host-plant-mediated speciation in the diversification of phytophagous insect 58 lineages is also largely emphasized in the literature on large-scale patterns of insect diversity: 59 macroevolutionary perspectives on phytophagous insect evolution have attributed their 60 extraordinary diversification to selective responses to their host-plants (Ehrlich & Raven 1964;

1 See glossary 2

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61 Janz 2011; Winkler & Mitter 2008; Yokoyama 1995). However, these macroevolutionary 62 scenarios are often presented in the literature as narratives for specific lineages commenting a 63 phylogenetic reconstruction of the history of host-plant associations. Many phylogenetic studies 64 still fail to clearly formulate hypotheses and predictions about the speciation processes that 65 underlie the observed patterns and the role played by host-plant adaptation in those. The reason 66 might be that the macroevolutionary patterns that arise when host-plant specialization* is the 67 driver of speciation events are not always clear. There is no review on what to expect and how to 68 formally test these predictions. 69 The increasing availability of robust molecular phylogenies and recent developments in 70 phylogenetic comparative methods are offering an unprecedented opportunity to test evolutionary 71 hypotheses in a robust phylogenetic framework. Our purpose here is to present the 72 macroevolutionary scenarios for the diversification of phytophagous insects that have been put 73 forward in the literature, decipher the role that ecological speciation driven by host-plant 74 adaptation play in them and synthetize predictions from these scenarios. We then identify tools 75 from the “comparative phylogenetic toolbox” that provide ways to test some of these predictions. 76 This toolbox can be divided into three compartments: 77 1) comparisons of the phylogenetic histories of insects and their associated plants: the 78 congruence (in terms of dates of divergence and branching patterns and) of the phylogenetic 79 histories of plant-feeding insects and their host-plants can be tested in robust statistical 80 frameworks and illuminate how herbivores track the diversification of their hosts; 81 2) ancestral character state reconstructions: the evolutionary trajectory of host-associations, 82 host breadth and host-plant adapted traits can be inferred using ancestral character state 83 reconstruction methods and statistical tests can determine whether their distribution throughout the 84 phylogenetic trees follow the predictions of scenarios involving ecological speciation mediated by 85 host-plant adaptation; 86 3) diversification analyses: the recent developments of methods to study the diversification 87 dynamics* of entire clades using phylogenetic trees provide ways to test how shifts to new host- 88 plant species or changes in host breadth have impacted speciation rates in phytophagous insects.

89 We review papers that have adopted these approaches. We then present suggestions for future 90 research that should help linking microevolutionary studies on host-plant adaptation and 91 macroevolutionary perspectives on phytophagous insect diversification.

92 I Macroevolutionary scenarios of phytophagous insect diversification

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93 I.1) Escape and radiate (Figure 1 a) 94 More than 50 years ago, Ehrlich & Raven (1964) put forward a macroevolutionary 95 scenario that inspired most of the current research on plant-feeding insect diversification: it is 96 known as “Escape and radiate” (Thompson 1989). They hypothesized that when insects acquire 97 the ability to circumvent the chemical defenses of a plant group, it promotes their rapid 98 diversification by ecological release, i.e. the availability of novel resources and reduction in direct 99 competition. Insects undergo an adaptive radiation*. In this scenario, adaptation towards host- 100 plants is the driving force of insect species formation. The “Escape and radiate” scenario also 101 hypothesizes that, in response to phytophagous insect predation, plants acquire novel chemical 102 defenses which allow them in turn to diversify very rapidly (Marquis et al. 2016). Ehrlich & 103 Raven’ s seminal study suffers from several shortcomings that have been pinpointed before (Janz 104 2011). First, although the authors frame their theory within the concept of adaptive radiation, they 105 do not explicitly lay out speciation mechanisms for both partners of the interaction. Following 106 their scenario, a trade-off in resource use and specialization towards specific host-plants is 107 necessary to explain the formation of numerous insect species (i.e. species radiation) following the 108 capture of a new host-plant lineage. Such a trade-off is not mentioned in the original paper (Janz 109 2011). In addition, as underlined by contemporary researchers of Erlich and Raven’s, it is difficult 110 to conceive how the selection pressures exerted by insects on plant defences can drive plant 111 speciation (Jermy 1976, Jermy 1984). Plant traits that reduce phytophagous insect attacks are 112 rarely linked with reproductive isolation between plant populations (but see Marquis et al. 2016 113 for a review of scenarios of herbivore-induced speciation in plants) and the evidence for bursts of 114 speciation in plants following the evolution of chemical defence is scant (Futuyma & Agrawal 115 2009). However this study has been and remains a great source of inspiration for studies on the 116 diversification of plant-insect associations. This is probably because it is one of the first studies 117 that attempts to explain how microevolutionary processes (host-plant adaptation) translate into 118 macroevolutionary patterns (radiation onto newly acquired plant lineages). Several predictions that 119 can be tested on phylogenetic trees arise from the Escape & Radiate scenario (Table 1). 120 In the years following its publication, “Escape and radiate” was often interpreted as 121 generating cospeciation* patterns; however it is now recognized that it rather predicts the 122 sequential speciation of insects onto an already diversified plant lineage (Janz 2011; Suchan & 123 Alvarez 2015). According to this prediction: 1) the reconstruction of the history of host-plant 124 associations on the phylogenetic trees of insects should reveal host-plant conservatism, i.e. the use 125 related plant species by related insects (Mitter & Brooks 1983; Winkler & Mitter 2008); 2) the 126 phylogenies of herbivorous insects and their host-plants should be more congruent than expected

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127 by chance and the diversification of the insects should lag behind that of their host-plants; this is 128 called sequential evolution (Jermy 1976) or “host tracking”. Nevertheless, when the association 129 between insects and their host plants is species-specific, a pattern of cospeciation can be expected 130 through simple co-vicariance: geographic barriers affect the differentiation of populations of 131 interacting lineages in a similar way and cause simultaneous speciation events (Althoff et al. 2012; 132 Brookes et al. 2015; Martínez-Aquino 2016). In these cases, it is geographic isolation and not 133 natural selection that initiates the reproductive isolation of insect populations and subsequent 134 speciation. However, the specificity of the insects and host-adapted traits enhance the probability 135 of shared vicariant events. 136 The diversification dynamics of insects should follow the typical pattern of adaptive 137 radiations (Janz 2011), i.e. they should show an acceleration of speciation rate upon the capture of 138 new plant lineages or the evolution of detoxification mechanisms (Wheat et al. 2007) and then 139 slow down when their niches are saturated (when species diversity is reaching the carrying 140 capacity of the host-plant lineage) (Rabosky & Lovette 2008). Furthermore, the capture of a 141 species-rich clade of plants should result in higher speciation rates than the capture of lineages 142 encompassing less species (Roskam 1985; Janz et al. 2006).

143 I.2) The ‘Oscillation Hypothesis’ (Figure 1b)

144 The “Escape and radiate” scenario was revisited more than a decade ago by Janz and 145 collaborators (Janz et al. 2001, Janz & Nylin 2008; Janz et al. 2006; Nylin & Janz 2009). Using 146 butterflies as study systems, they stated that expansions in diet breadth followed by specialisation 147 onto new host-plant species constantly fuel the diversification of phytophagous insects. This has 148 been termed the ‘Oscillation Hypothesis’ (Janz & Nylin 2008). It stipulates that transitions 149 towards a generalist diet generally open up a new adaptive zone, which favours the capture of new 150 host-plants. In this scenario, expansions in diet are enabled by the phenotypic plasticity of insects 151 with respect to host-plants (Nylin & Janz 2009). Population fragmentation and their specialisation 152 onto newly captured host-plants then lead to the formation of new species. Hence this scenario 153 explicitly predicts that species formation results from insect populations evolving towards the 154 utilization of a restricted set of host-plants. Nevertheless, it suggests that this specialization 155 process is often a consequence of the geographic isolation of generalist insect populations in areas 156 inhabited by different host-plant species (Janz et al. 2006). Therefore the “Oscillation hypothesis” 157 does not necessarily postulate that natural selection is the main driving force of species formation.

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158

159

160 c 161

162 Figur 1: Schematic illustrations of the macroevolutionary scenarios of phytophagous insect 163 diversification.

164

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165 However, subsequent papers quoting this scenario emphasize the central role of host-plant 166 specialisation (Hardy & Otto 2014; Nakadai 2017; Wang et al. 2017; Hardy 2017). 167 Most of the predictions from the Escape and Radiate scenario are valid under the 168 “Oscillation hypothesis” (Hardy et al. 2017), however it yields several new predictions (Table 1): 169 - generalist* diets should be “transient and repeatedly disappear in favour of specialization 170 onto a limited set of related plants” (Nylin et al. 2014); 171 - gains of new host plants are associated with host breadth expansion (Janz et al. 2001); 172 - the amplitude of the oscillation determines the number of potential host-plant species; 173 therefore, insect clades with the most diverse host-use (the highest number of host-plant species) 174 are expected to be more speciose than clades using less host species (Janz et al. 2006); and along 175 the same lines insect clades that encompass species that exhibit large host breadths should have 176 higher diversification rates (Hardy & Otto 2014; Weingartner et al. 2006); 177 - shifts from a generalist diet to a specialist one should be associated with an acceleration 178 of diversification rates. In other words, patterns of diversification should follow a model where 179 cladogenetic events are associated with host breadth reduction (Hardy & Otto 2014); 180 - generalists have larger geographic ranges as they are able to colonize more habitats and 181 can expand more easily (Slove & Janz 2011).

182 I.3) The Musical Chairs (Figure 1c)

183 Hardy and Otto (2014) have recently put forward a scenario where speciation in 184 herbivorous insects is driven by specialisation onto newly captured host-plants without any 185 transitions in diet breadth: insects speciate by successive switches to new host-plants. The authors 186 named their scenario “the Musical Chairs” (Hardy & Otto 2014). They explicitly state that 187 specialization onto a few host plant species explains species diversification in phytophagous 188 insect. 189 The “musical chairs” hypothesis yields several predictions that differentiate it from the 190 “Oscillation hypothesis” (Table 1): 191 - gains of new hosts are not associated with host breath expansion (Hardy 2017): 192 - host breadth contraction is not associated with cladogenetic events (Hardy & Otto 2014); 193 - speciation rates should be higher in lineages showing no conservatism in host-plant 194 associations (Hardy & Otto 2014); 195 - lineages showing many transitions from generalist to specialist feeding diets should not 196 be more speciose than lineages that only encompass specialist species (Hardy & Otto 2014).

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197 However, several authors have pointed out that it will be difficult to tell apart the “Musical 198 Chairs” from “the Oscillation Hypothesis”. If generalism is indeed ephemeral as expected when 199 specialization towards host-plants is adaptive, it will be difficult to reconstruct its history 200 accurately on phylogenetic trees (Janz et al. 2016). Consequently, the relationships between host- 201 range size and speciation rates will be difficult to explore and the set of predictions that 202 differentiate the musical chairs from the oscillation hypothesis will not always be testable. 203 Phylogenetic comparative methods (Pennell & Harmon 2013) including increasingly 204 sophisticated diversification models (Beaulieu & O’Meara 2016; O’Meara & Beaulieu 2016; 205 Rabosky 2006; Rabosky & Goldberg 2015; Stadler 2013; Stadler & Bokma 2013) can now be 206 deployed to reconstruct ancestral character states, investigate the diversification dynamics of 207 insect lineages and test whether shifts in diversification rates are associated with transitions in 208 character states. Below we review how these methods have been used to investigate the evolution 209 of plant/insect associations and test the predictions of host-driven speciation scenarios.

210 II Phylogenetic approaches for testing ecological speciation scenarios

211 II.1) Comparing the phylogenies of plants and insects

212 Many phylogenetic studies have compared the phylogenies of herbivorous insect and their 213 host-plants and have investigated cospeciation and host tracking patterns. They have used tools 214 such as tree reconciliation analyses (Conow et al. 2010; Page 1994) and distance-based methods 215 for tree comparisons (Legendre et al. 2002). A pattern of cospeciation has been found between 216 figs and their phytophagous pollinating wasps (Cruaud et al. 2012; Rønsted et al. 2005; Weiblen 217 & Bush 2002) as predicted by early taxonomic studies on this biological system (Ramirez 1974; 218 Wiebes 1979), but also between figs and some of the non-pollinating galling wasps that are highly 219 specific to their hosts (Jousselin et al. 2008). Reciprocal adaptations of plants and insects (i.e. 220 coevolution*) have been unravelled in this study system (Jousselin et al. 2003; Weiblen 2004). 221 However, it is not known whether the cospeciation patterns observed are the sole result of this 222 coevolution (i.e. whether reciprocal selection exerted by both partners has driven the reproductive 223 isolation of interacting populations; Althoff et al. 2014; Hembry et al. 2014) or whether matching 224 speciation events have arisen through co-vicariance. In the other iconic model system for the study 225 of plant/insect coevolutionary diversification*, the Yucca–Yucca moth interaction (Pellmyr 226 2003), pollinating and non-pollinating moths phylogenetic histories parallel some parts of the 227 evolutionary history of their hosts. Some studies suggest that these patterns are the results of 228 coevolution (Godsoe et al. 2009) while others hint towards co-vicariance (Althoff et al. 2012)..

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229 Table 1: Predictions from the different scenarios involving host-plant driven speciation: the first column indicates the evolutionary hypotheses tested

230 and the headers of the other columns indicate phylogenetic comparative approaches used for testing them.

Insect and host tree Evolution of host Evolution of host breadth Insect diversification dynamics comparison associations Speciation driven by Cospeciation if the insects play No overlap in host use Predominance of specialists over - herbivorous insects are more diverse than their host-plant a role in their host-plant among sister-species generalists (Janz et al. 2001, non-herbivorous relatives (Mitter et al. 1988) specialization reproductive isolation (Nyman et al. 2010). Winkler & Mitter 2008). - the diversification dynamic of phytophagous insects follows a pattern of adaptive radiation (Janz 2011) - the number of species within an insect clade positively correlates with the number of host- plant species (Janz 2006) Macroevolutionary scenarios Escape and radiate Host tracking (Jermy 1976, Phylogenetic No prediction Increase in speciation rates upon the capture of Mitter & Brooks 1983). conservatism of host- new plant lineages or detoxification mechanisms plant lineages or host- (Wheat et al. 2007: Fordyce 2010). plants with similar defences (Winkler & Mitter 2008). Oscillation Potentially host tracking Conservatism of host- - recurrent transitions in host - clades including generalist species are more following the capture of a new plants following the breadth (Janz et al. 2001, Janz & speciose than clades with only specialists host-plant lineage. capture of a new plant Nylin 2008) (Weingartner et al. 2006) lineage. - gain of new host lineages - speciation rates higher in lineages showing high preceded by host breadth expansion lability in host breadth (Hardy & Otto 2014) (Janz et al. 2001) - host breadth larger in species with large geographic range (Slove & Janz 2011) - speciation events associated with shifts from generalist to specialist (Hardy & Otto 2014) Musical chairs No prediction High lability in speciose Few transitions in host breadth - no positive association between speciation rates lineages breadth (Hardy (Hardy & Otto 2014; Hardy 2017) and host breadth lability (Hardy & Otto 2014) & Otto 2014) - speciation rates positively correlated with lability in host associations (Hardy & Otto 2014)

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231 In both interactions, the fact that the phytophagous insects are specific pollinators of their 232 host-plants and breed inside them necessarily link the reproductive success of the two partners 233 and increase the likelihood of concomitant speciation events. Therefore host-plant adapted 234 traits are certainly pivotal in the speciation process of these insects. 235 Several studies have unravelled a pattern of phylogenetic tracking between 236 phytophagous insects and their host-plants (e. g. Althoff et al. 2006; Becerra & Venable 1999; 237 Farrell & Mitter 1990; Farrell & Mitter 1998; Miller et al. 1987; Percy et al. 2004; Roskam 238 1985) suggesting that phytophagous insects in different orders (Coleoptera, Lepidoptera, 239 Hemiptera) have speciated by switching and specialising onto different subsets of a newly 240 captured plant lineage, partly mimicking their host-plant phylogenies. All these studies give 241 credit to the Escape and Radiate scenario. 242 In many case studies, the phylogenies of plants and insect groups were not 243 simultaneously available or the patterns of host associations precluded any possibility of 244 phylogenetic tracking. The authors have then simply compared the timing of divergence of 245 plants and associated insects (e. g. Brandle et al. 2005; Kergoat et al. 2011; Kergoat et al. 246 2015; Lopez-Vaamonde et al. 2006; McKenna et al. 2009; McLeish et al. 2013; Pena & 247 Wahlberg 2008; Segar et al. 2012; Stone et al. 2009; Vea & Grimaldi 2016; Wahlberg et al. 248 2013). Most of these studies suggest delayed (but sometimes rapid) colonization of already 249 diversified groups of plants by insects groups at different temporal scales. They are generally 250 based on mere qualitative comparisons of dates of divergence obtained from fossil calibrated 251 phylogenies of both plants and insects, but can also include thorough statistical comparisons 252 of dates obtained through phylogenetic methods (Loss-Oliveira et al. 2012; McLeish et al. 253 2013). They are generally framed as supporting the Escape and radiate theory. However these 254 studies do not give any information on the speciation process behind the diversification of the 255 insect lineages studied; they merely indicate the timing of host plant colonization. The 256 comparison of the diversification dynamics* of both herbivorous insects and their host-plants 257 provide a more direct test of host-driven speciation hypotheses: under host-driven adaptive 258 radiation insects diversification dynamic is expected to roughly follow the diversification of 259 its host- plant lineage (Kergoat et al. 2018) (see II. 3..2). 260 261 II. 2) Reconstructing the evolutionary trajectory of traits involved in host-plant use 262 II.2.1) Evolution of host associations 263 Phylogenetic inferences are now widely used to reconstruct the evolutionary 264 trajectories of phenotypic traits throughout the diversification of a lineage. Most phylogenetic

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265 studies of phytophagous insects map the history of host association onto the resulting trees. 266 These reconstructions often show host conservatism (see Winkler & Mitter 2008 for a 267 review). These assertions stem from mere observations of the reconstructions but numerous 268 studies now include statistical tests. These include the permutation tail probability test (PTP, 269 Faith & Cranston 1991- e. g. Kelley and Farrell 1998), or some index of phylogenetic signal 270 such as the lambda (ʎ) of Pagel (1999) (see for instance Leppanen et al. 2012; Lopez- 271 Vaamonde et al. 2003; Wilson et al. 2012 for statistical evidence of host conservatism on

272 respectively: sawflies, leaf-mining moths and geometrid moths). 273 Host conservatism is often interpreted as following the predictions of “Escape and 274 Radiate” and therefore evidence that speciation was promoted by host-plant specialization. 275 However, showing that related insects feed on related plants does not say much about the 276 process that has generated this pattern nor connects mechanistically host-plant use evolution 277 to speciation. The use of vague wording such as host-associations favour or constrain 278 speciation is commonly found when discussing host-conservatism in the literature and it is 279 difficult to actually conclude from these studies that specialization towards one or a few 280 plants species is the main pathway towards the formation of new phytophagous insect species. 281 The pattern of “host conservatism” is in agreement with a scenario in which insects have 282 radiated onto a plant lineage but it could also suggest that host-plant shifts are not important 283 promoters of speciation events. 284 A more direct estimation of the contribution of host-plant adaptation in the speciation 285 process consists in inferring the frequency of host-plant shifts in relation to speciation events. 286 If adaptation to different ranges of host-plants drives reproductive isolation and speciation, it 287 follows that insect sister species should partition host-plant resources: i.e. they should show 288 no or little overlap in the plant species they use ( Nyman et al. 2010). 289 To investigate factors driving speciation in a genus of bark beetles (Aphanarthrum), 290 Jordal & Hewitt ( 2004) simply compared host use of sister species* and estimated that only 291 two out of twelve cladogenetic events could be associated with host shifts. Nyman et al. 292 (2010) used a phylogeny of sawflies belonging to the Nematinae (Hymenoptera) and 293 reconstructed the evolution of their ecological niches (defined as the combination of feeding 294 habits and host-plant families). They showed that the number of niche shifts represented at the 295 most 60% of the number of speciation events. They also demonstrated that the probability that 296 sister species overlapped in their niche decreased with time since the speciation event, 297 suggesting that more recently diverged species have more chance of sharing host-plant 298 species and thus have probably not differentiated via host shifts. Jousselin et al. (2013)

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299 adopted a similar approach on a genus of conifer-feeding aphids (Hemiptera). Species in this 300 genus generally feed on one or few species and ecological niches were defined as a 301 combination of “plant organ x host-plant species”. They showed that the number of niche 302 shifts observed only represented 20% of the speciation events and was lower than expected if 303 the niches were randomized onto the phylogeny; suggesting that ecological speciation was not 304 the main process behind the diversification of this aphid lineage. Recently Winkler et al. 305 (2018) showed that species splits associated with niche differentiation (host-plant species 306 and/or type of plant tissue attacked) were less numerous than geographic shifts throughout the 307 phylogeny of a genus of tropical fruit-flies (Blepharoneura). To quantity niche overlap at 308 cladogenetic events, Nakadai & Kawatika ( 2016) used an approach that resemble Disparity 309 Through Time studies (Harmon et al. 2003). They computed a dissimilarity index that 310 calculates the difference in host use across the different nodes of the phylogeny and tested 311 whether closely related species share a more similar range of host-plant than expected by 312 chance. They show that changes in host-plant use are concentrated at the root of the tree and 313 play a minor role in recent speciation events. 314 Linnen et al. (2010) adopted yet a different approach to investigate the role of host 315 shifts in speciation. They suggested that if host-shifts triggered speciation events, the 316 evolution of host association should follow a speciational model of evolution, in which 317 changes in host use occur during speciation events and its probability is not related to branch 318 length. They thus compared the likelihood of a speciational vs a gradual model of evolution 319 on a phylogeny of Neodiprion (Hymenoptera) and demonstrated that the speciational model 320 was more likely, implying that host shifts accompany speciation events. 321 Hence, the studies that estimated niche differentiation at speciation events gave mixed 322 support for scenarios where specialization onto different sets of host-plants is the main 323 speciation process. It is nevertheless surprising that such studies have not been conducted on 324 more study systems. This is perhaps due to the fact that they require a precise knowledge of 325 the range of host-plants used by each insect species.

326 II.2.2) Host-breadth evolution 327 In order to test the predictions of macroevolutionary scenarios and investigate the role 328 of specialization in insect species formation, phylogenetic studies have also investigated how 329 host breadth is distributed throughout the evolutionary history of insects groups. 330 According to the “Oscillation hypothesis”, host breadth should vary a lot along the 331 phylogeny of insects and the character state “generalist” should be transient (Nylin et al.

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332 2014); i.e. the phylogenetic reconstruction of this character should show many transitions 333 between specialists and generalists. Kelley & Farrell (1998), using ancestral character state 334 reconstructions, have shown that host-breaths were indeed labile in Dendoctronus beetles and 335 that specialists could evolve towards generalists. Several studies have then estimated the rates 336 of transition between specialists and generalists. Using the phylogenies of fifteen insect 337 genera from various orders, Nosil & Mooers (2005) estimated that the transition rates toward 338 specialization exceed the transition rates toward generalization and that specialization was not 339 a dead-end. More recently, Simonsen et al. (2010) reported repeated broadenings of diet in a 340 genus of butterflies but did not quantify the phylogenetic signal of host breadth. Janz et al. 341 (2001) and Nylin et al. (2014) have demonstrated the lability of host breadths in Nymphalids 342 using indicators of phylogenetic signal and Wang et al. (2017) also showed that this character 343 was highly labile in moths. 344 These patterns suggest that changes in host breadths are recurrent in the evolutionary 345 history of insect lineages and are therefore compatible with the Oscillation hypothesis. In 346 order to test whether these changes are linked with the colonization of new hosts (favouring 347 subsequent specialization and speciation), Janz et al. (2001) inferred the number of gains and 348 losses of host plants throughout the history of Nymphalini. They found that gains exceeded 349 losses and suggested that these were the result of repeated range expansions. However Hardy 350 (2017) recently reanalysed the same dataset using different models of evolution of host use 351 and inferred equal numbers of gains and losses of plants throughout the phylogeny of 352 Nymphalini. He concluded that in this insect group host shifts and speciation events do not 353 necessarily result from host range expansions and contractions. May be another way to 354 investigate the link between ‘host-breadth changes’ and ‘capture of new hosts’ would be to 355 investigate correlated evolution of these two characters. In any case, the recent debates around 356 the oscillation hypothesis (its predictions and how to test them) clearly demonstrates that 357 ancestral character state reconstructions and statistical tests based on these reconstructions are 358 very sensitive to model choices and character coding strategies and should always be 359 interpreted with care, even when very specific predictions are verified.

360 II.2.3) Evolution of host-plant adapted traits 361 Many studies that aimed at finding support for the coevolutionary arm race 362 hypothesized by the Escape and Radiate scenario have investigated the evolution of 363 detoxification mechanisms in insect lineages and how they correlate with changes in host 364 associations. Becerra (1997; 2003) reconstructed the evolution of detoxification mechanisms

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365 and plant defence in a genus of Chrysomelidae and their associated host plants (Burseraceae), 366 and showed synchronous evolution of defences and counter defences that agree with a 367 scenario of host-plant driven radiation in these insects. Similarly, in a review on the 368 interaction between the chemically-defended Heliconius butterflies and their Passiflora host- 369 plants, de Castro et al. (2018) highlighted a large variety of anti-herbivory adaptations in the 370 plants (including chemical defences, trichomes, fake eggs or herbivore damages, interaction 371 with ants) and counter-adaptations in the butterflies that support a long history of coevolution. 372 Also in agreement with the Escape and Radiate theory, Wahlberg (2001) using 373 ancestral character states reconstruction on a phylogeny of Melitaeini (Lepidoptera) showed 374 that host switches occurred between plants with similar defences, rather than closely related 375 plants (i.e. host-plant use was more influenced by chemistry than by plant taxonomy). Endara 376 et al. (2017) showed opportunistic host switches to plants with similar defences in a genus of 377 butterfly. They suggested that herbivores "choose" host-plants based on their own defensive 378 traits. The authors conclude that disruptive selection is not a driver of speciation in this case. 379 Many other studies looked at the diversification dynamics of genes involved in plant 380 detoxification and how it correlates with changes in host-plant associations. Wheat et al. 381 (2007) showed that the evolution of a detoxification mechanism, a nitrile-specifier protein 382 (NSP) in Pieridae matches the distribution of glucosinolate in their host-plants. Edger et al. 383 (2015) further investigated arm races between Brassicales and butterflies (Pieridae) and 384 showed that repeated evolution of nitrile-specifier proteins were associated with bursts of 385 diversification over the past 80 Myr in Pieridae. Calla et al. (2017) examined the cytochrome 386 P450 monooxygenase (CYP) gene superfamily diversification in the genomes of seven 387 Lepidoptera species varying in host breadth. They showed that its dynamics (duplication and 388 losses) was correlated with the ability to metabolise defences. Bramer et al. (2015) analysed 389 the ability to sequester toxic cardenolides throughout the phylogeny of the hemipteran 390 subfamily Lygaeinae and suggested that it was acquired in response to selection by 391 cardenolide-producing Apocynaceae host-plants. 392 Reconstruction of the history of plant defences were also conducted on plant 393 phylogenies. For instance, Livshultz et al. (2018) reconstructed the evolution of cardenolide 394 production in Apocynaceae and suggested that it could have evolved in response to

395 herbivorous insect predation. On the other hand, Agrawal et al. (2009), reconstructed the 396 history of plant defences in North American milkweed species (Asclepias, Apocynaceae) and 397 showed that less investment in cardenolide production correlates with an increase in 398 speciation rates. This does not follow the predictions of “Escape and Radiate” and rather

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399 suggest that investment in costly defences might have impeded diversification in this plant 400 group. 401 In contrast to detoxification mechanisms, traits (and underlying genes) involved in 402 host recognition and host-plant choice (chemosensory traits) have been less studied in a 403 phylogenetic context. However their evolutionary dynamic probably plays as important a role 404 as adaptations to plant defences in phytophagous insect speciation (Smadja & Butlin 2009). 405 Matsuo (2008) showed that an odour binding protein in 27 Drosophila species can evolve 406 relatively fast in closely related species through gene duplications and losses, and proposed 407 that this dynamic could explain the evolution of host preferences in this species complex. 408 Sánchez-Gracia et al. (2009) and Vieira & Rozas (2011) conducted a comparative genomic 409 analysis of odour binding protein and chemosensory proteins from the genomes of several 410 Arthropoda species (mainly Drosophila). They showed a high number of gains and losses of 411 genes, pseudogenes, and independent origins of gene subfamilies. This dynamic if analysed in 412 relation to host choices and host breadth in a phylogenetic context could explain some host 413 shifts and subsequent speciation events. 414 Focusing on behavioural traits of insects, Molnar et al. (2018) analysed the antennal 415 responses of 12 gall midge species to a wide range of host-plant-related volatiles and showed 416 that species with similar response shared host-plants. They therefore suggested that 417 modification of olfaction is associated with host-shifts and speciation in this system. 418 Finally deciphering the evolutionary dynamics of genes involved in mate recognition 419 and their link with host association could also inform us on the role of host-plants in the 420 speciation of insects that feed (and often mate) on them. For instance, Schwander et al. (2013) 421 showed that cuticular hydrocarbon profiles involved in mate choices vary among Timema 422 species (Coleoptera), and that most evolutionary change in hydrocarbon profiles occurs in 423 association with host-plant shifts and speciation events in this genus of phytophagous insects. 424 This study shows that physiological traits involved in reproductive isolation can be associated 425 with host plant differentiation on a macroevolutionary time scale. 426 In summary, many studies have investigated the evolution of detoxification 427 mechanisms in butterflies and found support for Escape and Radiate. More studies 428 investigating changes in traits involved in host recognition throughout the diversification of 429 insects are need in order to determine which traits underlie host-plant associations and 430 whether their evolution accompany speciation events (see Perspectives).

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431 II. 3) Studying how host-plants use impact the diversification dynamics of herbivorous 432 insects

433 Methods for testing diversification dynamics have expanded over the last decade. 434 Testing for the existence of temporal bursts of diversification was once restricted to analyses 435 of groups with comprehensive fossil records. Diversification dynamics can now be studied 436 through comprehensive phylogenies (Goswami et al. 2016). Given the breadth of available 437 methods, theoretically, nearly all hypotheses can be put to test.

438 II.3.1) Are phytophagous insects more diversified than their related counterparts? 439 Studies that posit that host-plant adaptation favours phytophagous insect 440 diversification predict that those are much more diversified than non-phytophagous insects. 441 However this assertion deserves to be statistically tested. The first studies addressing this 442 question (Farrell & Mitter 1990; Farrell & Mitter 1998; Mitter et al. 1988), compared the 443 diversity (number of species) of phytophagous vs related non-phytophagous clades in beetles. 444 They all suggested that herbivorous clades are more diverse than their non-phytophagous 445 sister clades. However, Hunt et al. (2007) and then Rainford et al. (2015), using more 446 comprehensive beetle phylogenies, and also adopting a sister clade comparison, did not find a 447 significant increase in species richness in phytophagous clades. In their phylogeny of the 448 beetle family Erotylidae, Leschen & Buckley (2007) mapped the evolution of species number 449 within clades (as a two-state character: 0/1) and did not detect any significant correlation 450 between species richness and phytophagy (using Bayesian tests of character correlation). 451 McKenna et al. (2015) tested for temporal variation in diversification rates (using MEDUSA 452 Alfaro et al. 2009) on a global phylogeny of Coleoptera and showed that those underwent 453 several accelerations of diversification rates; some seemed to be associated with the evolution 454 of phytophagy while others were not. 455 There are more informative tests than those that merely test whether one group 456 contains more species or has diversified more rapidly than others. More recently Wiens et al. 457 (2015) used Phylogenetic Generalized Least Square Regressions and comprehensive 458 phylogenies within and between several insect orders and showed that the proportion of 459 phytophagous insect species in a clade was positively correlated with diversification rates in 460 several insect orders. But this relationship did not hold for Coleoptera, Hymenoptera and 461 Orthoptera. Likelihood-based character state dependent diversification models (known as, the 462 –SSE models, such as BiSSE, ClaSSE and QuaSSE for binary, multistate and quantitative 463 traits, respectively FitzJohn 2010; Maddison & FitzJohn 2015; Maddison et al. 2007) can

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464 specifically test whether transition in character states are associated with variations in 465 speciation and extinction rates. These diversification models can be used to test whether the 466 evolution of phytophagy has favoured diversification. However, they were not employed in 467 Wiens et al. (2015) as a robust estimate of diversification parameters by these methods would 468 require dense and random species sampling throughout the phylogeny. According to FitzJohn 469 (2010) more than 15% of the species must be included in the phylogeny in order to conduct 470 unbiased –SSE tests.The study was conducted at the order level and within each order the 471 proportion of missing species was too high to apply –SSE methods. 472 From current studies using more comprehensive phylogenies and statistical tests, it is 473 thus yet not completely clear whether phytophagy increases diversification rates in insects in 474 comparison to other life habits. The availability of more comprehensive phylogenies will 475 allow testing this hypothesis on more insect orders using character state dependent 476 diversification models. Nevertheless; a caveat of these diversification models is that they may 477 overlook more complex models involving many unmeasured and co-distributed traits 478 (especially when few transitions in feeding habits are observed). The HiSSE model (Beaulieu 479 & O’Meara 2016) which models hidden characters that might influence diversification might 480 help untangling these confounding factors.

481 II.3.2) Testing for adaptive radiation 482 Diversification analyses can also be applied to test whether the capture of new host- 483 plants has favoured adaptive radiations. Under such a scenario, the diversification curve of 484 phytophagous insect clades should exhibit early bursts of speciation upon the capture of new 485 groups of host-plants. Eventually, the lineages are expected to ﬁll the newly found niche 486 space and the diversification curves should reach a plateau. 487 There are several studies that have observed acceleration in diversification rates in 488 insect lineages and have put forward ad-hoc narratives that attribute this acceleration to the 489 capture of new host-plant lineages or detoxification mechanism. For instance, Wheat et al 490 (2007) showed that the Pierinae clade that can detoxify glucosinolate in their host plants are 491 more diversified that its sister clade. Mullen et al. (2011) have observed an increase in species 492 richness in the butterfly genus Adelpha (Nymphalidae) and attributed this increase to adaptive 493 divergence in response to host-plant diversity found within the neotropics (namely host-plant 494 shift to Rubiaceae and other plant families). Ebel et al. (2015) revealed an increase in the rates 495 of diversification on the phylogeny of the genus Adelpha and observed that those were 496 concomitant to host shifts. Sahoo et al. (2017) revealed two accelerations in diversification in

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497 skipper butterflies that they attributed to shifts from dicots to monocots at time where those 498 diversified and expanded. Fordyce (2010) showed changed in diversification rates upon the 499 capture of new plant lineages in several butterfly lineages and Edger et al. (2015) detected 500 shifts in diversification rate associated with the colonization of new host-plants with new 501 defences, though this was not statistically tested. Winkler et al. (2018) on the other hand, 502 uncovered stable diversification rates and no decrease associated with overlap in host use 503 throughout the phylogeny of the fly genus Blepharoneura, suggesting that this lineage did not 504 undergo an adaptive radiation.Using BiSSE, Peňa & Espeland (2015) found that a hostplant 505 shift to Solanaceae was correlated with an increase in net diversification rates in Ithomiini 506 butterflies (Nymphalidae). However, since only one shift to Solanaceae occurred in the 507 Nymphalidae, this correlation should not be taken as evidence that hostplant shift has driven 508 diversification of this butterfly tribe. Instead, the radiation of Ithomiini may be linked with 509 geographical context in this study. 510 While several studies have uncovered acceleration in diversification rates upon the 511 capture of new host-plant lineages, only a handful have investigated whether the number of 512 species reaches a plateau after an initial burst of diversification. Meseguer et al. (2015) 513 studied the diversification dynamic of a conifer-feeding aphid genus. They revealed an 514 accelerated rate upon the capture of conifers but no saturation as expected under a scenario of 515 adaptive radiation. The use of density-dependent models of diversiﬁcation would be better 516 suited to test whether rates of diversiﬁcation decrease through time during evolutionary 517 radiations (Etienne et al. 2012; Rabosky & Lovette 2008). But these tests can only be applied 518 to lineages in which the number of species in each subclade is well known. Peňa et al. (2015) 519 used such diversity-dependent birth-death models to infer speciation, extinction rates and 520 carrying capacity on a phylogenetic tree of the butterfly genus Erebia and showed that the 521 diversification dynamics was consistent with a model of adaptive radiation. Kergoat et al. 522 (2018) compared the diversification dynamics of Sesamiina stemborer moths and their 523 associated grasses. The initial burst of diversification observed in moths suggests that their 524 emergence might have been favoured by the diversification of their host-plants. A decline in 525 speciation rates was then inferred in these insects using Diversity-dependent birth-death 526 models. However, their host-plants continued to thrive. This challenges the “adaptive 527 radiation scenario” and suggests that these moth diversification patterns cannot solely be 528 explained but the availability of suitable host-plants (Kergoat et al. 2018). 529 530 II.3.3) Correlating host breadth with diversification dynamic

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531 Advocates of the Oscillation hypothesis suggest that clades showing a higher diversity 532 of host-use (using more host-plant species altogether) should be more diverse than their sister 533 clade (Janz et al. 2006), and this prediction has been verified in butterflies. Janz et al. (2006) 534 and Nylin & Wahlberg (2008) found a positive relationship between species diversity of 535 butterflies and species diversity of host-plant taxa in several genera of Nymphalidae, through 536 a sister clade comparisons of number of species. Joy & Crespi (2012) and Lin et al. (2015) 537 found a similar result in respectively gall-inducing flies and Coccidae. Wang et al. (2017) 538 used a sister clade comparisons and PGLS to demonstrate a similar trend in moths. 539 Although these results support a model where the diversity of phytophagous insects is 540 sustained by the diversity of the hosts they use, they could fit both a model of Oscillation and 541 the Musical chairs hypothesis. The latter indeed predicts that lineages including specialist 542 species that often switch between hosts, use a large number of host-plants. To tell apart the 543 Oscillation from the Musical chairs, it is actually more informative to test how host breadth 544 variations affect insect diversification dynamics. According to the Oscillation hypothesis, 545 clades including generalist species should be more speciose than clades including only 546 specialists. Weingartner et al. (2006) tested this prediction in Polygonia butterflies 547 (Nymphalidae) through sister clade analyses and showed that clades using a broader host- 548 plant range are more species-rich than their sister group that encompass species that only use 549 the ancestral hosts (here, urticalean rosids), in agreement with the Oscillation scenario. 550 More finely tuned analyses, such as trait-dependant diversification models (BiSSE and 551 QuaSSE models) have been used on the phylogenies of Papillionidaea and the tribe 552 Heliconiini by Hardy and Otto (2014). In this paper, where the Musical chairs hypothesis 553 hwas put forward, the authors showed that: 1) speciation rates decreased with host breadth 554 (i.e. monophagous species diversified faster than polyphagous species); 2) changes in host 555 breadth were not associated with cladogenetic events; 3) rates of host switching were 556 positively associated with diversification rates. All these results were in agreement with the 557 Musical chair hypothesis. Still on Nymphalidae, (Hamm & Fordyce 2015) found that host 558 breadth characteristics were phylogenetically conserved which does not fit the predictions of 559 the Oscillation hypothesis about the lability of host-breadth and also found that speciation 560 rates did not increase with host-breadth expansion, in agreement with the Musical chair 561 scenario. 562 By contrast, Hardy et al. (2016) showed on a Coccidea phylogeny that host breadth 563 (measured as the number of host-plant families) was positively correlated to speciation rates. 564 As –SSE models are known to generate false positives (Bouchenak-Khelladi et al.

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565 2015;Rabosky & Goldberg 2015; Davis et al. 2013), they conducted the analysis on a set of 566 randomized trees in order to test whether the constrained diversification model (i.e. the model 567 in which evolutionary transitions in character states are associated with shifts in extinction 568 and/or speciation rates) is also chosen in these analyses. The authors did not frame their study 569 within the Oscillation vs Musical chairs controversy, but if we refer to the paper of Hardy & 570 Otto (2014), these results fit with some of the predictions of the Oscillation hypothesis. 571 As already mentioned, there are debates on the influence of host breadth variation on 572 diversification dynamics (Hamm & Fordyce 2015; Janz et al. 2016; Hamm & Fordyce 2016; 573 Wang et al. 2017). The transient nature of the generalist feeding diet under host-driven 574 speciation scenarios makes it difficult to derive clear predictions (Janz et al. 2016). 575 Furthermore, if specialization towards host plants can accelerate speciation rates it can also 576 increase extinction risks when plants are not highly abundant. Although diversification 577 methods can potentially differentiate extinction rates from speciation rates (Morlon 2014), 578 predicting exactly how changes in host-plant breadth affects diversification dynamics remains 579 difficult. 580 Finally, many studies have been conducted on butterflies. In order to have a better 581 understanding of how phytophagous insects capture new host-plants and whether it influences 582 their diversification dynamics, it seems necessary to test the predictions of macroevolutionary 583 scenarios on other insect groups. Aphids could be good candidates for such investigation. 584 Their range of host-plants is very well documented (Blackman & Eastop 2006; Holman 585 2009). Although most aphids are host-specific, there are some polyphagous species. Some 586 species are even only polyphagous during the asexual part of their life-cycle (Jousselin et al. 587 2010; Moran 1992; Hardy et al. 2015). This temporary broadening of diet has already been 588 suggested to facilitate the capture of new host-plants (Moran 1992) and subsequent 589 specialization and speciation on these new hosts. These life-cycle y transitions could have 590 thus favoured the diversification on this group (Jousselin et al. 2010; Moran 1992). This 591 scenario fits the Oscillation hypothesis and should be tested on a robust and comprehensive 592 aphid phylogeny. Other insect groups for which host plant repertoire are well known such as 593 Coccidae (Garcia-Morales et al. 2016) and Psyllids (Ouvrard et al. 2015) could also be used 594 to test the predictions of macroevolutionary scenarios.

595 In summary, diversification analyses have been widely applied to phytophagous 596 insects. These methods were fairly basic in early papers but they have been refined throughout 597 the years and their use allowed reframing hypotheses on the role of host-plant specialization

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598 on insect speciation. Recent results brought mixed evidence for phytophagy as an accelerator 599 of diversification. Trait-dependant diversification analyses supported alternative scenarios 600 involving oscillation in diet breadths as a driver of host-plants shifts and speciation, and from 601 our review, it is likely that different scenarios will prevail in different lineages and even 602 probably in the same lineage at different time scales. 603

604 III Perspectives 605 As seen throughout this review, phylogenetic comparative methods provide the 606 template to test hypotheses on the role of host plant association in the speciation of 607 phytophagous insects. While those methods have undoubtedly advanced the field significantly 608 since “the Escape and Radiate” paper, readers must keep in mind that phylogenetic 609 comparative methods often rely on mere correlations. Significant associations between 610 character changes and the cladogenetic events might arise as a consequence of speciation 611 itself when post-speciational character changes occur. Furthermore, comparisons of models of 612 evolution such as those used in trait-dependent diversification analyses often rely on trees that 613 encompass few transitions in character states and are therefore not always robust. In such 614 analyses, the “best model” is not necessarily the true model and significant P values should 615 not be interpreted as strong evidence for an evolutionary scenario. Finally, all these methods 616 are very sensitive to sampling biases and those need to be carefully taken into consideration. 617 In addition to using the approaches focused on host plant associations and diet breadth 618 cited throughout this review, one way to further investigate hypotheses of speciation driven 619 by associations with host-plants would be to integrate a variety of data in a phylogenetic 620 context. Below we outline three potential directions for future research: 1) disentangling the 621 role of plant-insect interaction from that of co-variates, such as geography and climate; 2) 622 combining phylogenetic analyses with interaction network approaches including other 623 partners, at various ecological scales (from community-scale to global scale); and 3) studying 624 traits and genes underlying the association.

625 III.1) Investigating the role of abiotic factors: geography and climate

626 Geography and ecology are always closely intertwined in speciation scenarios. There have 627 been several studies that have investigated geographic range expansion in herbivorous insects 628 (Becerra & Venable 1999; Slove & Janz 2011); climate induced host shifts (see Nyman et al. 629 2012 for a review and recent studies since then Lisa De-Silva, 2017; Owen et al. 2017; 630 Pitteloud et al. 2017; Sahoo et al. 2017; Sanchez-Guillen et al. 2016; Winkler et al. 2009) and

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631 climate driven diversification dynamics (Kergoat et al. 2018). All these studies suggest that 632 abiotic factors are entangled with host-plants changes in species diversification scenarios. 633 However there are few studies that explicitly test the predictions of speciation through 634 geographic isolation (Barraclough & Vogler 2000) and whether these events systematically 635 accompany host shifts or sustain most speciation events (but see Jordal & Hewitt 2004; 636 Jousselin et al. 2013; Doorenweerd et al. 2015; Hardy et al. 2016). Such analyses are 637 important if we want to tell whether adaptations to new host plants represent post-speciational 638 changes following geographic isolation rather than the main driver of speciation events. 639 Cospeciation methods that take into account the biogeographic history of interacting lineages 640 (Berry et al. 2018) could be also used to investigate whether host shifts are associated with 641 dispersal events in systems where hosts plants and insects phylogenies show some congruent 642 patterns. 643

644 III.2) Combining phylogenetic with interaction network approaches, at various 645 ecological scales

646 Herbivores and the plants they feed on form interaction networks, and as such the 647 structures of the networks can be characterized by several parameters, such as modularity (the 648 propensity of a group of species to interact with a similar set of partners) and nestedness (the 649 propensity of specialist species to interact with generalist species and vice-versa). 650 Antagonistic interaction networks, such as plant-herbivore networks, tend to be highly 651 modular (Thébault & Fontaine 2010). A recent study combining interaction network with 652 phylogenetic approaches on simulated and real datasets predicted that the Escape and radiate 653 scenario should produce a modular network structure, whereas the Oscillations scenario 654 should produce a more nested structure (Braga et al. 2018). When applied to real data (two 655 butterfly families, Nymphalidae and Pieridae), this approach revealed that host-plant butterfly 656 networks tend to be both modular and nested, which the authors interpret as being the result of 657 a complex pattern of diversification, involving both episodes of radiation on new hosts 658 (producing modules containing closely related species) and occasional shifts to other host 659 lineages, producing both nestedness within modules and connections between modules. 660 Additionally, phylogenetic and network approaches could be expanded to encompass other 661 interacting partners (e. g. Elias et al. 2013, Ives & Godfray 2006) . Indeed, insect-host-plant 662 communities can be seen as ecosystems where biotic interactions, such as parasitism and 663 mutualism also take place (Forister et al. 2012). These other partners can indirectly influence

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664 the interaction between plants and there herbivores: e. g., direct competition (Jermy 1988) 665 apparent competition between herbivores, stemming from shared natural enemies, (Holt 666 1977), and vice-versa (e. g., when herbivory elicits anti-herbivore defences mediated by 667 herbivore enemies, (Fatouros et al. 2008)). Multitrophic interactions probably explain many

668 diversification patterns in herbivorous insects (Singer & Stireman 2005) 669 Finally, such approaches could be applied both at a large scale (e. g., Braga et al. 2018), to 670 embrace global patterns of diversification and interaction, or at the community level (Elias et 671 al. 2013, Ives & Godfray 2006), where interactions actually occur, and where fine-scale 672 processes (e. g., host-plant shift at the species or the population level) can be unveiled.

673 III.3) Unravelling traits involved in the interaction and their underlying genes, and 674 integrating this information in phylogenetic studies 675 Interactions between insects and their host-plant are ultimately mediated by traits, such as 676 host-plant defences and the capacity of circumventing plants defences, but also host-plant 677 cues and the capacity for herbivores to detect those cues. Characterizing such traits, their 678 genetic determinism and looking at their evolutionary trajectory would greatly advance our 679 understanding of the diversification of insects (e. g., de Castro et al. 2018). In addition, 680 methods that test whether patterns of trait evolution conform to a model accounting for 681 interactions mediated by those traits are currently being developed (Manceau et al. 2016; 682 Drury et al. 2017), and they could inform on the processes underlying herbivore 683 diversification. However, targeting traits involved in plant-insect interaction may be 684 challenging. Pivotal traits are difficult to identify, they include chemical, behavioral and 685 metabolic traits and when they are properly characterized they are often multigenic. 686 Perhaps a promising direction for future research is the implementation of a hybrid 687 genomic approach that combines transcriptomics, phylogenomics, comparative analyses and 688 population genomics (see Nevado et al. 2016 for an example on plants). In such approaches, 689 full transcriptomes of species from a target clade (for instance, a clade of phytophagous 690 insect) are generated. These transcriptomes (or other sequence data) are used to generate a 691 phylogeny, where classical diversification and character evolution tests can be performed 692 (evolution of characters, test for diversity-dependent diversification, shifts in diversification 693 following host-plant shift). Then, genes under selection can be detected from transcriptomic 694 data using population genetics statistics, and can be matched to existing databases (e. g., 695 Lepbase for Lepidoptera, Challis et al. 2016) for identification purpose. Additionally, genes 696 that are down or upregulated can also been detected by classical tests of differential

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697 expression and identified, and the association of genes under selection, either via different 698 sequence or expression pattern, with species diversity can be tested. The main limits of this 699 approach lie in the availability of specimens (transcriptomic data need to be obtained from 700 fresh or suitably preserved tissues; biological replicates are needed), and the quality of the 701 reference gene database to match genes with putative functions. 702

703 Conclusions

704 Many phylogenetic studies of plant-insect associations now include formal tests of 705 macroevolutionary scenarios involving host-driven speciation. In an attempt to summarize the 706 literature on this topic, we show that the predictions of host-plant driven speciation are not 707 straightforward and can vary depending on studies. We advocate a standardization of these 708 predictions to facilitate cross study analyses. Furthermore, it is also recognized that different 709 scenarios can leave the same phylogenetic signature (Janz et al. 2016) and that depending on 710 the analytical approaches undertaken to test the predictions laid out in Table 1, conclusions 711 can vary (Hardy et al. 2017). Unfortunately this means that the interpretations of phylogenetic 712 inferences can remain somewhat subjective. But these shortcomings should not obscure the 713 progresses that have been made in the field. Phylogenetic comparative analyses help framing 714 hypotheses and clarify some of the narratives used to explain the diversification of 715 phytophagous insects. Finally, this survey of the literature shows that: 1) the simple 716 assumption that phytophagy has accelerated insect diversification is not always sustained by 717 meta-analyses; 2) the expectation that sister lineage will use different ranges of host plants is 718 not often tested, and, when it is, the predictions of a host-driven speciation scenarios are not 719 always met. We then underline that the results of phylogenetic comparative methods cannot 720 be interpreted as hard evidence as they remain mere correlations. In the end, a full 721 understanding of the processes explaining the diversification of phytophagous insects will 722 require the integration of phylogenies with other data sources and analytical methods. We 723 propose here a few perspectives to integrate such data and investigate host-driven speciation 724 scenarios on a macroevolutionary time scale.

725 Finally, if the last two decades have seen the rise of molecular phylogenies and the 726 development of analytical methods that include ecological data, this should not obscure the 727 fact that this data needs to be thoroughly cured before any phylogenetic comparative analyses. 728 Qualifying host associations of insect species necessitates field work and advanced taxonomy, 729 as mistakes can seriously impact the results of macroevolutionary studies. Functional studies

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730 aimed at deciphering host-plant adapted traits in insects (and in particular traits implied in 731 host choice) and characterizing genes that underlie these traits are also needed to integrate this 732 data in a phylogenetic context and link microevolutionary processes with macroevolutionary 733 scenarios.

734 Glossary:

735 Adaptive radiation: the evolution of ecological and phenotypic diversity within a rapidly 736 multiplying lineage. It occurs when natural selection drives divergence of an ancestral species 737 into descendants that exploit different ecological niches. 738 739 Coevolution: reciprocal evolutionary changes occurring in two or more species that result 740 from reciprocal selective pressures exerted by the interacting partners.

741 Coevolutionary diversification: when diversification patterns arise from coevolution.

742 Cospeciation: simultaneous speciation events in lineages involved in long-term interspecific 743 associations which result in congruent phylogenies

744 Diversification dynamic: rates of species formation and extinction through time.

745 Ecological specialization: when species limit themselves to a restricted set of ressources 746 (diet- habitat-niches), as a result of evolutionary trade-offs.

747 Generalists: species that use a wide niche.

748 Host-plant adaptation: heritable trait that confers a selective advantage on a particular host- 749 plant.

750 Specialists: species that use a narrow niche.

751 Phytophagous insect: an insect that feeds on any plant organ during whole or part of its life 752 cycle, it excludes pollinators feeding on nectar and pollen but include pollinators that feed on 753 developing seeds (i.e. seminiphagous insects).

754 Sister species/ sister clades: the closest relatives of another given unit (species/ clade) in a 755 phylogenetic tree.

756

757 Conflict of interest disclosure

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758 The authors of this preprint declare that they have no financial conflict of interest with 759 the content of this article."

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767 Althoff DM, Segraves KA, Johnson MTJ (2014) Testing for coevolutionary diversification: linking 768 pattern with process. Trends in Ecology & Evolution 29, 82-89.

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